Progesterone-dependent changes in platelet activation without morphological variation in diestrus mares

Progesterone (P4) exerts important vascular and immunomodulatory effects that influence platelet (PLT) activation and serotonin (5-HT) handling across mammalian species; nevertheless, its role in modulating PLT physiology during diestrus in mares remains poorly defined. This study hypothesized that physiological variations in luteal activity during diestrus are associated with changes in PLT activation and 5-HT-related parameters. The first objective was to determine whether changes in circulating P4 during diestrus are associated with alterations in PLT aggregation, circulating 5-HT, and PLT morphological indices in healthy mares; the second objective was to identify a diestrus day providing consistent physiological conditions for assessing PLT-related biomarkers. Twenty clinically healthy Spanish Purebred mares aged 4–9 years old were monitored. Blood samples were collected on days 5, 14, and 16 post-ovulation, with luteal status confirmed by ultrasonography. P4 concentrations were determined using a solid-phase I-125 radioimmunoassay (RIA), 5-HT concentrations were quantified using a competitive enzyme immunoassay, and PLT indices were measured using an ADVIA 2120i hematology analyzer. Data were compared using appropriate parametric or non-parametric tests after assessing distribution, and correlations were analyzed using rank-based correlation analysis, using Pearson or Spearman coefficients according to variable distribution. P4 concentrations were higher on days 14 and 16 compared with day 5 (p < 0.05), with no significant differences between days 14 and 16. Platelet aggregates (AGREG) showed the greatest variation, with significantly higher values on day 14 compared with day 5 (p < 0.05). In contrast, circulating 5-HT and all PLT morphological indices (PLT count, PCT, MPV, PLCR, PDW, PCDW, MPM, and PMDW) remained unchanged across diestrus. PLT aggregation showed a strong positive association with circulating P4 concentrations (r = 0.88, p < 0.05), whereas no meaningful correlations were observed between 5-HT and AGREG or between 5-HT and PLT morphological parameters. Internal correlations among PLT indices followed expected biological patterns, confirming the stability of structural PLT traits over short physiological intervals. These findings demonstrate that during diestrus, PLT activation—but not PLT morphology or circulating 5-HT—varies in parallel with P4 in mares. Day 14, corresponding to mid-diestrus, characterized by high luteal activity, represents an informative time point for assessing PLT activation and related biomarkers, providing a framework for standardizing sampling protocols for PLT-derived products in equine reproductive medicine.